Role of CEBPb in flow-dependent endothelial dysfunction and atherosclerosis

CEBPb 在血流依赖性内皮功能障碍和动脉粥样硬化中的作用

• 批准号: 10638650
• 负责人: Hanjoong Jo
• 金额: $ 75.4万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10638650
• 关键词: Acute; Address; Animal Model; Antiatherogenic; Antibodies; Aorta; Arteries; Atherosclerosis; Binding; Biological Assay; Blood flow; CCRL2 gene; Cells; Chromatin; Chronic; Coronary; Coronary artery; Data; Data Set; Development; Diet; Disease; Embryo; Endothelial Cells; Endothelium; Exposure to; Fibroblasts; Gene Expression Regulation; Genes; Genome; Human; Immune; Inflammation; Inflammatory; Knowledge; Left; Length; Ligation; Literature; Mesenchymal; Messenger RNA; Metabolic; Methods; Modeling; Mus; Myocardial Infarction; Nuclear; Open Reading Frames; Pathway interactions; Peripheral arterial disease; Phenotype; Plasmids; Production; Protein Isoforms; Proteins; Proteomics; Regulation; Role; Small Interfering RNA; Stains; Stroke; Tertiary Protein Structure; Testing; Therapeutic; Time; Transcript; Translating; Translation Initiation; Translations; atherogenesis; cancer type; carcinogenesis; chronic inflammatory disease; design; endothelial dysfunction; epigenome; epigenomics; expression vector; hypercholesterolemia; imaging modality; in vivo; inhibitor; interest; multiple omics; mutant; new technology; novel; overexpression; single-cell RNA sequencing; transcription factor; transcriptome; transcriptomics; ultrasound; western diet; Acute; Address; Animal Model; Antiatherogenic; Antibodies; Aorta; Arteries; Atherosclerosis; Binding; Biological Assay; Blood flow; CCRL2 gene; Cells; Chromatin; Chronic; Coronary; Coronary artery; Data; Data Set; Development; Diet; Disease; Embryo; Endothelial Cells; Endothelium; Exposure to; Fibroblasts; Gene Expression Regulation; Genes; Genome; Human; Immune; Inflammation; Inflammatory; Knowledge; Left; Length; Ligation; Literature; Mesenchymal; Messenger RNA; Metabolic; Methods; Modeling; Mus; Myocardial Infarction; Nuclear; Open Reading Frames; Pathway interactions; Peripheral arterial disease; Phenotype; Plasmids; Production; Protein Isoforms; Proteins; Proteomics; Regulation; Role; Small Interfering RNA; Stains; Stroke; Tertiary Protein Structure; Testing; Therapeutic; Time; Transcript; Translating; Translation Initiation; Translations; atherogenesis; cancer type; carcinogenesis; chronic inflammatory disease; design; endothelial dysfunction; epigenome; epigenomics; expression vector; hypercholesterolemia; imaging modality; in vivo; inhibitor; interest; multiple omics; mutant; new technology; novel; overexpression; single-cell RNA sequencing; transcription factor; transcriptome; transcriptomics; ultrasound; western diet

Atherosclerosis is a chronic inflammatory disease and preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow) while those in the stable flow (s-flow) regions are protected. The mechanisms by which d-flow and s-flow regulate atherogenesis are still not well-understood. To address this critical knowledge gap, we recently conducted a scRNAseq and a scATACseq assay using the mouse partial carotid ligation (PCL) model of atherosclerosis caused by d-flow. The study revealed that d-flow dramatically alters endothelial transcriptome and epigenomic profiles, reprogramming them into the inflammatory, mesenchymal (EndMT), and immune cell-like (EndICLT) phenotypes, which we defined as “endothelial reprogramming (EndRep)”. While the pro-atherogenic role of inflammation and EndMT is well-established, the mechanisms and role of EndICLT and EndRep in atherosclerosis are unknown. We identified CEBPb through a reanalysis of our scRNAseq and scATACseq datasets as a candidate transcription factor that could regulate EndRep. Our preliminary data further show that the expression of CEBPb increased by d-flow in vivo and cultured HAECs. CEBPb produces three different protein isoforms, LAP1, LAP2, and LIP, through alternative translation. Surprisingly, CEBPb is translated primarily as the LIP protein in HAECs. Further, d-flow stimulates the nuclear localization of LIP in HAECs, and overexpression of LIP dramatically induces EndRep (endothelial inflammation, EndMT, and EndICLT). Our proteomics study shows that LIP binds the PSMB9 immunoproteasome protein, and d-flow- increases the PSMB9 activity. Furthermore, prior studies showed that LIP induces cancer-type metabolic reprogramming. Therefore, we hypothesize that d-flow stimulates nuclear expression of LIP, which binds to PSMB9, leading to EndRep and atherosclerosis. We will test the hypothesis with three aims. Aim 1 will determine the effect of d-flow on LIP nuclear expression and its role in EndRep. EC-specific-Confetti mice will be used for lineage tracing to validate the flow regulation of EndICLT and EndRep. HAECs treated with si-CEBPb or LIP plasmid and mice with EC-specific LIP overexpression (LIPEC-OE) or LIP deficiency (LIPDEF) will be used. An ultrasound-guided method will be used to deliver LIP to the left carotid of LIPDEF mice to induce EndRep. Aim 2 will determine how LIP induces EndRep in ECs flow-dependent manner. scRNAseq & scATACseq assays will be conducted using the LIPEC-OE and LIPDEF mice with the PCL to identify LIP and flow-regulated genes/pathways. Flow-regulation of PSMB9 activity and its role in LIP-induced EndRep will be determined in HAECs and mice using siRNAs and PSMB9 inhibitors. Aim 3 will determine the role of LIP in atherosclerosis in a flow- and PSMB9- dependent manner. Atherosclerosis in LIPEC-OE and LIPDEF will be studied in the acute PCL and chronic model using AAV-PCSK9 and western diet with or without PSMB9 inhibitor treatment. scRNAseq and scATACseq analysis will be conducted in these mice. Human coronary staining for CEBPb will determine its pathophysiological significance. These studies could reveal potential anti-atherogenic therapeutic avenues.

动脉粥样硬化是一种慢性炎症性疾病，优先发生在暴露于 稳定流动（S-Flow）区域中的血流受到干扰（D-Flow）受到保护。这些机制 D-Flow和S-Flow调节动脉粥样硬化仍然没有得到充分理解。解决这一批判知识 差距，我们最近使用小鼠部分颈动脉连接（PCL）进行了SCRNASEQ和SCATACSEQ分析 由D-Flow引起的动脉粥样硬化模型。研究表明，d-flow极大地改变了内皮 转录组和表观基因组谱，将其重新编程为炎症，间质（EndMT）和 免疫细胞样（Endiclt）表型，我们将其定义为“内皮重编程（EndRep）”。而 炎症和末端的亲震源作用是良好的，Endiclt和Endiclt和 动脉粥样硬化中的终结尚不清楚。我们通过重新分析我们的scrnaseq，并确定了CEBPB SCATACSEQ数据集是可以调节Endrep的候选转录因子。我们的初步数据 表明cebpb的表达通过体内和培养的HAEC增加了D-Flow。 CEBPB生产三个 通过替代翻译，不同的蛋白质同工型，LAP1，LAP2和LIP。令人惊讶的是，CEBPB是 在HAEC中主要翻译为唇部蛋白。此外，d-flow刺激嘴唇的核定位置 HAEC和唇部的过表达极大地诱导了Endrep（内皮炎症，EndMT和 Endiclt）。我们的蛋白质组学研究表明，唇部结合PSMB9免疫蛋白酶体蛋白，D-Flow-- 增加PSMB9活性。此外，先前的研究表明，唇部影响癌症代谢 重新编程。因此，我们假设D-Flow刺激了嘴唇的核表达，该唇与 PSMB9，导致终结和动脉粥样硬化。我们将以三个目标检验假设。 AIM 1将确定 D-Flow对唇部核表达及其在Endrep中的作用的影响。 EC特异性小鼠将用于 谱系跟踪以验证Endiclt和Endrep的流量调节。用SI-CEBPB或嘴唇处理的HAEC 将使用具有EC特异性唇部过表达（LIPEC-OE）或唇部缺乏症（LIPDEF）的质粒和小鼠。一个 超声引导的方法将用于向Lipdef小鼠的左颈椎输送唇部，以诱导Endrep。目标2 将确定唇部如何以ECS流动依赖性方式诱导Endrep。 scrnaseq＆scatacseq分析将 使用PCL使用LIPEC-OE和LIPDEF小鼠进行识别唇部和流动调节的基因/途径。 PSMB9活性的流动调节及其在唇部诱导的终结中的作用将在HAEC和小鼠中确定 使用siRNA和PSMB9抑制剂。 AIM 3将确定唇部在流动和PSMB9-的动脉粥样硬化中的作用 依赖方式。 LIPEC-OE和LIPDEF中的动脉粥样硬化将在急性PCL和慢性模型中研究 使用AAV-PCSK9和西方饮食进行或不接受PSMB9抑制剂治疗。 scrnaseq和scatacseq 分析将在这些小鼠中进行。 CEBPB的人类冠状动脉染色将确定其 病理生理意义。这些研究可以揭示潜在的抗动脉本体治疗途径。